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The Metabolism of 1,8-Dinitropyrene by Salmonella typhimuriumOrr, Joan 09 1900 (has links)
S. typhimurium strain TA98/1,8-DNP₆ is resistant to mutagenesis by 1,8-dinitropyrene and is deficient in an acetyl-CoA dependent acetyltransferase activity. Strains TA98 and TA98NR which are acetyltransferase competent are sensitive to 1,8-dinitropyrene mutagenesis. The coincidence of acetyltransferase deficiency and 1,8-dinitropyrene resistance in strain TA98/1,8-DNP₆ implicates acetylation as an important process in the metabolic activation of 1,8-dinitropyrene to a mutagenic intermediate. The acetyltransferase activity can be assayed by observing the formation of 1-N-acetylamino-8-aminopyrene and 1,8-N,N'-diacetyldiaminopyrene from 1,8-diaminopyrene. Reduction of the nitro-function is also an important enzymatic step involved in the activation of 1,8-dinitropyrene. Evidence is presented which suggests that a nitroreductase-acetyltransferase enzyme complex may exist. Further investigations of nitroreductase activity have yielded results which indicate that three distinct nitroreductases exist, one specific for 1,8-dinitropyrene, one specific for 1-nitropyrene and one specific for nitrofurazone. The implications of these findings are discussed and an explanatory model is proposed. / Thesis / Master of Science (MS)
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Estudio de inocuidad de Salmonella entérica, subespecie entérica, serotipo Enteritidis, var Danysz lisina negativa en pollos parrillerosRodríguez Ayala, Natalia Karina Soledad January 2005 (has links)
Se realizó un estudio con la cepa Salmonella enterica, subespecie entérica, serotipo Enteritidis, var. Danysz lisina negativa, principio activo de un producto comercial usado para el control biológico de roedores. Evaluándose su inocuidad en pollos parrilleros. Se usaron 120 aves de 1 día de edad de la linea Cobb Vantress divididas en 3 grupos, con igual porcentaje de hembras y machos. El grupo A fue alimentado los días 5, 6 y 7 de edad con un alimento comercial que contenía 20% del producto raticida. El grupo B fue inoculado al 8vo día de edad directamente al buche con 1 ml. de 108 UFC de la bacteria. El tercer grupo permaneció como control. Se registraron signos clínicos y mortalidad diaria, peso corporal individual semanal, recuperación de la cepa de Salmonella usada y reapuesta serológica. A partir del quinto día postinoculación y durante una semana, dos aves de cada grupo fueron necropsiadas. Adicionalmente se tomaron muestras de sangre a todos los grupos a los 21, 28 y 35 días post inoculación y en aves sacrificadas, para detectar anticuerpos a Salmonella mediante la prueba de aglutinación en placa. Los resultados del estudio bacteriológico, y de las pruebas serológicas realizadas entre el día 5 a 12 post desafio fueron negativos a Salmonella spp. No se encontraron lesiones macroscópicas compatibles con infección por Salmonella, sin embargo a los 28 y 35 días post desafio las aves del grupo A dieron 5.5% a los 28 días (1/18) y 16.6% a los 35 días de edad (3/18) aves positivas a la prueba de aglutinación para Salmonella spp.. las aves que reaccionaron serológicamente fueron sometidas a una evaluación bacteriológica, se aisló Salmonella spp. tipificada como lisina positiva. Los pesos corporales a la sétima semana no mostraron diferencias significativas entre grupos. / The Salmonella enterica serotipe Enteritidis var Danysz negative lysine strain is used as a component of a product for biological control of rodents. This product is a mixture of grains containing the strain. The present study evaluated whether the strain was innocuous for chickens or not. One hundred and twenty day – old Cobb Vantress broiler chicks (50% each sex) were divided into 3 groups of 40 chicks each. Four chicks per group were separated to verify if they were free from Salmonella spp. The group A was fed with standard broilers food containing 20% of the product for biological rodent control. The group B received 1 ml containing 108 CFU of Salmonella enterica serotipe Enteritidis var Danysz negative lysine by crop gavage at eighth day. The group C was kept as uninoculated control group. Clinical signs, mortality, recovery strain used in the experiment, body weight, and serological response were recorded. Chicks were observed daily for clinical signs and mortality. Bacteriological cultures were made in order to recover the microorganism from liver and spleen. All birds were weighed every week. Two chicks were randomly selected from each treatment group, euthanatized, and necropsied since the fifth day during one week. Serum samples and select tissues (liver and spleen) were collected for bacteriological culture. Additionally, serum samples were collected from each group in the 21st , 28th and 35th days postinoculation. The results of bacteriological work of select tissues (direct and indirect cultures) and the serological study of serum samples tested negative for Salmonella spp. Positive birds to the agglutination test to Salmonella spp. were found only in the group A. It was found 5.5% at 28 days and 16.6% at 35 days of age. The positive birds were submitted to a bacteriological evaluation; in one of them Salmonella spp was isolated and typified by biochemistry as positive lysine. Significant weight differences among groups recorded in the seventh week, were not found.
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Estudio de inocuidad de Salmonella entérica, subespecie entérica, serotipo Enteritidis, var Danysz lisina negativa en pollos parrillerosRodríguez Ayala, Natalia Karina Soledad January 2006 (has links)
Se realizó un estudio con la cepa Salmonella enterica, subespecie entérica, serotipo Enteritidis, var. Danysz lisina negativa, principio activo de un producto comercial usado para el control biológico de roedores. Evaluándose su inocuidad en pollos parrilleros. Se usaron 120 aves de 1 día de edad de la linea Cobb Vantress divididas en 3 grupos, con igual porcentaje de hembras y machos. El grupo A fue alimentado los días 5, 6 y 7 de edad con un alimento comercial que contenía 20% del producto raticida. El grupo B fue inoculado al 8vo día de edad directamente al buche con 1 ml. de 108 UFC de la bacteria. El tercer grupo permaneció como control. Se registraron signos clínicos y mortalidad diaria, peso corporal individual semanal, recuperación de la cepa de Salmonella usada y reapuesta serológica. A partir del quinto día postinoculación y durante una semana, dos aves de cada grupo fueron necropsiadas. Adicionalmente se tomaron muestras de sangre a todos los grupos a los 21, 28 y 35 días post inoculación y en aves sacrificadas, para detectar anticuerpos a Salmonella mediante la prueba de aglutinación en placa. Los resultados del estudio bacteriológico, y de las pruebas serológicas realizadas entre el día 5 a 12 post desafio fueron negativos a Salmonella spp. No se encontraron lesiones macroscópicas compatibles con infección por Salmonella, sin embargo a los 28 y 35 días post desafio las aves del grupo A dieron 5.5% a los 28 días (1/18) y 16.6% a los 35 días de edad (3/18) aves positivas a la prueba de aglutinación para Salmonella spp.. las aves que reaccionaron serológicamente fueron sometidas a una evaluación bacteriológica, se aisló Salmonella spp. tipificada como lisina positiva. Los pesos corporales a la sétima semana no mostraron diferencias significativas entre grupos. / The Salmonella enterica serotipe Enteritidis var Danysz negative lysine strain is used as a component of a product for biological control of rodents. This product is a mixture of grains containing the strain. The present study evaluated whether the strain was innocuous for chickens or not. One hundred and twenty day – old Cobb Vantress broiler chicks (50% each sex) were divided into 3 groups of 40 chicks each. Four chicks per group were separated to verify if they were free from Salmonella spp. The group A was fed with standard broilers food containing 20% of the product for biological rodent control. The group B received 1 ml containing 108 CFU of Salmonella enterica serotipe Enteritidis var Danysz negative lysine by crop gavage at eighth day. The group C was kept as uninoculated control group. Clinical signs, mortality, recovery strain used in the experiment, body weight, and serological response were recorded. Chicks were observed daily for clinical signs and mortality. Bacteriological cultures were made in order to recover the microorganism from liver and spleen. All birds were weighed every week. Two chicks were randomly selected from each treatment group, euthanatized, and necropsied since the fifth day during one week. Serum samples and select tissues (liver and spleen) were collected for bacteriological culture. Additionally, serum samples were collected from each group in the 21st , 28th and 35th days postinoculation. The results of bacteriological work of select tissues (direct and indirect cultures) and the serological study of serum samples tested negative for Salmonella spp. Positive birds to the agglutination test to Salmonella spp. were found only in the group A. It was found 5.5% at 28 days and 16.6% at 35 days of age. The positive birds were submitted to a bacteriological evaluation; in one of them Salmonella spp was isolated and typified by biochemistry as positive lysine. Significant weight differences among groups recorded in the seventh week, were not found.
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Drug resistance and R-plasmids in salmonellae in Hong Kong /Ling, Mei-lun, Julia, January 1985 (has links)
Thesis (Ph. D.)--University of Hong Kong, 1986.
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A study of the carrier state in Salmonella infectionDavies, Rodney January 1975 (has links)
xiv, 207 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1976
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A study of the carrier state in Salmonella infection.Davies, Rodney. January 1975 (has links) (PDF)
Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 1976.
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Characterization of the type VI protein secretion system encoded in the Salmonella pathogenicity island 19 and its role in the pathogenicity of serotypes Gallinarum and EnteritidisBlondel Buijuy, Carlos José January 2011 (has links)
Thesis Submitted in Partial Fulfillment for the Requirements to
achieve the Degree of PhD in Biochemistry / El genero Salmonella comprende a mas 2,500 serotipos conocidos distribuidos
en dos especies: enterica y bongori. Estos serotipos difieren mucho en términos de
patogenicidad y especificidad hospedero. Dos serotipos de Salmonella entérica son de
especial relevancia: los serotipos Gallinarum y Enteritidis.
S. Gallinarum presenta un rango hospedero restringido a aves y causa una
severa enfermedad sistémica conocida como tifoidea aviar, la que causa grandes
perdidas económicas en la producción aviar en distintas partes del mundo. S.
Enteritidis, en cambio, infecta a un amplio rango de hospederos incluyendo humanos,
ratones y aves. A diferencia de S. Gallinarum, S. Enteritidis genera una infección
subclinica en los pollos, y las aves infectadas pueden convertirse en portadores
crónicos, poniendo huevos contaminados por Salmonella. El consumo humano de
productos aviares o huevos resulta en un cuadro de gastroenteritis aguda autolimitante,
la cual es responsable por ~61% del 1.5 millones de casos de salmonelosis reportados
entre los años 1995 y 2008 (WHO Global Foodborne Infections Network Country
Databank).
Existen pocos trabajos realizados sobre los mecanismos moleculares detrás de
la adaptación al hospedero aviar y sobre las implicancias clínicas de las infecciones
causadas por los serotipos Enteritidis y Gallinarum, sin embargo evidencia reciente
sugiere que estos serotipos poseen factores de virulencia no descritos que pueden ser
responsables de estas diferencias. La interación entre las bacterias y sus hospederos
es guiada por una comunicación dinámica que busca influenciar la respuesta del
hospedero. Dentro de las herramientas utilizadas por las bacterias para influir la
respuesta de sus hospederos, las maquinas secretoras que entregan proteínas y
toxinas hacia el ambiente intracelular de sus blancos eucariontes son cruciales para la
supervivencia y virulencia bacteriana. El Sistema de Secreción Tipo VI (T6SS) es un nuevo mecanismo de
translocación de proteínas que existe en la mayoría de bacterias Gram-negativo que se
encuentran en contacto íntimo con células eucariontes, incluyendo a aquellas que son
patógenos humanos y de plantas. El papel preciso que cumplen estos T6SS todavía es
desconocido pero es claro que cumple un papel importante en la virulencia bacteriana.
En Salmonella enterica, solo se ha descrito un T6SS el cual esta codificado en
la Isla de Patogenicidad 6 de Salmonella (SPI-6). En esta tesis, a través de análisis
bioinformaticos y de genomica comparativa se determinó que el genero Salmonella
codifica 5 T6SS, distribuidos diferencialmente entre distintos serotipos y con historias
evolutivas diferentes. Los nuevos T6SS fueron identificados en islas genómicas
designadas SPI-19, SPI-20, SPI-21 and SPI-22. Ademas de la identificación de estas
islas, una nueva proteína VgrG “evolucionada” con un dominio del tipo S-Piocina fue
identificado en SPI-21. La presencia de este dominio sugirió por primera vez un papel
de los T6SS en muerte bacteriana, abriendo un nuevo capitulo en el estudio de T6SS y
su papel en relaciones interbacterianas.
El T6SS de SPI-19 fue de especial relevancia debido a su amplia distribución
dentro de serotipos virulentos de Salmonella y porque análisis bioinformaticos
mostraron que mientras Gallinarum codifica un T6SS completo, el serotipo Enteritidis
solo codifica para remanentes de este sistema. A pesar de estar estrechamente
relacionados, los serotipos Gallinarum y Enteritidis presentan diferencias profundas en
su rango de hospederos y patogenicidad. Por lo tanto, es posible especular que la
presencia de un T6SS activo esta relacionada de alguna manera con las diferencias en
especificidad hospedero y patogenicidad presentada por estos dos serotipos. Para
resolver esta hipótesis y determinar la contribución de SPI-19 a la patogenicidad de
Salmonella, el objetivo de esta tesis fue determinar si la isla genomica SPI-19 codifica
un T6SS funcional que contribuye a la patogenicidad de Gallinarum y Enteritidis en el
hospedero aviar.
De manera de caracterizar el T6SS de SPI-19, fusiones génicas y de operon
fueron construidas y la expresión, producción y secreción de componentes del T6SS fueron evaluadas bajo diferentes condiciones de crecimiento in vitro. El análisis mostro
que la mayoría de los componentes se mantienen reprimidos bajo las condiciones
analizadas. Infección de macrófagos murinos con una cepa de Gallinarum con una
fusión entre el componente estructural/secretado VgrG al reportero GFP, mostró que
los componentes del T6SS son preferencialmente producidos al interior de células
infectadas. Mutantes por deleción no polares de la isla SPI-19 y componentes
específicos del T6SS reveló que este T6SS es necesario para la supervivencia de
Salmonella Gallinarum al interior de macrófagos a tiempos tardios de infección. Sin
embargo, el T6SS de SPI-19 no pudo ser asociado muerte celular o citotoxicidad de
macrófagos inducida por Salmonella.
Para determinar la contribución del T6SS de SPI-19 a la patogenicidad de
Salmonella, mutantes del T6SS fueron analizadas en ensayos de competencia contra
la cepa silvestre de Gallinarum. Infección oral de pollos White Leghorn de cuatro días
de edad, reveló que las mutantes del T6SS colonizaron pobremente el ileo, ciego,
hígado y bazo comparado a la cepa silvestre. Restitución de SPI-19 a la mutante SPI-
19, utilizando el sistema VEX-Capture, complementó este defecto en colonización.
Para analizar el impacto de poseer un T6SS completo en la habilidad de S. Enteritidis
para colonizar al hospedero aviar, la SPI-19 de Gallinarum fue transferida a Enteritidis.
Experimentos in vivo mostraron que la presencia de una SPI-19 completa aumento
significatvamente la habilidad de Enteritidis para colonizar el ileo, hígado y bazo de
pollos infectados al dia 1 post-infección. Sin embargo, esa ventaja en la colonización
no fue duradera ya que esta cepa mostró un fuerte defecto en la colonización desde el
día 3 post-infección hasta el final de los experimentos. Estos resultados sugieren que
transferencia de SPI-19 desde S. Gallinarum tiene un impacto negativo en la habilidad
de S. Enteritidis para colonizar el hospedero aviar. De esta forma podemos especular
que perdida del T6SS de SPI-19 corresponde a un evento patoadaptativo durante la
evolución de S. Enteritidis. Del mismo modo, este es el primer trabajo en el que se
utiliza el método VEX-Capture para determinar el efecto de la transferencia de islas
genómicas en un modelo animal de infección bacteriana. El reciente descubrimiento de Sistemas de Secreción Tipo VI (T6SS) ha abierto
un nuevo capitulo en el estudio de la adaptación de Salmonella hacia sus hospederos y
el medio ambiente. Si Salmonella codifica T6SS y si aquellos pueden ser considerados
eventos evolutivos cuanticos, fueron algunas de las preguntas que el descubrimiento
de los T6SS en los genomas bacterianos generó. En esta tesis, hemos expandido el
actual conocimiento sobre los T6SS bacterianos y el potencial patogénico de
Salmonella mediante: i) la identificación y descripción de 4 nuevas Islas de
Patogenicidad de Salmonella (SPI-19, SPI-20, SPI-21 and SPI-22) que codifican para
T6SS filogenéticamente distintos, ii) el descubrimiento de una nueva “VgrG”
evolucionada que sugirió por primera vez un papel de los T6SS en relaciones
interbacterianas, iii) identificando que el T6SS de SPI-19 contribuye a la supervivencia
intracelular de Salmonella en macrófagos y iv) determinando que el T6SS de SPI-19
contribuye a la colonización de pollos por S. Gallinarum. / The Salmonella genus includes over 2,500 known serotypes distributed between
the two species: enterica and bongori. These serotypes differ greatly in terms of
pathogenicity and host specificity. Two Salmonella enterica serotypes are of significant
relevance: serotypes Gallinarum and Enteritidis.
S. Gallinarum has a host range restricted to birds and causes a severe systemic
disease called fowl typhoid, which causes major economic losses in poultry production
in several parts of the world. S. Enteritidis on the other hand, infects a broad range of
hosts including humans, mice and avian species. In contrast to S. Gallinarum, S.
Enteritidis generates a subclinical infection in poultry, and infected hens can become
chronic carriers laying Salmonella contaminated eggs. Human consumption of
contaminated poultry or egg products results in an acute self-limiting gastroenteritis,
being responsible for ~61% of the estimated 1.5 million human salmonellosis cases
reported between 1995 and 2008 (WHO Global Foodborne Infections Network Country
Databank).
There is little work done on the molecular mechanisms behind the differential
host-adaptation and clinical outcomes of infections caused by serotypes Enteritidis and
Gallinarum in their susceptible hosts, including birds, but recent evidence suggests that
these serotypes might possess undescribed virulence factors that may account for
these differences. Interaction between bacteria and hosts is guided by a
communication/signaling interplay which aims to influence the host response. Among
the tools used by bacteria to influence the host response, secretion machines that
deliver proteins and toxins into the environment and within eukaryotic target cells are
crucial for bacterial virulence and survival.
The Type VI Secretion System (T6SS) is a newly described mechanism for
protein translocation that exists in most Gram-negative bacteria that come into close
contact with eukaryotic cells, including plant and animal pathogens. The precise role and mode of action of T6SS is still unknown, but it is clear that plays an important role
in bacterial virulence.
In Salmonella enterica, only one T6SS encoded in Salmonella Pathogenicity
Island 6 (SPI-6) has been described. In this thesis, through bioinformatics and
comparative genomic analyzes it was determined that the genus Salmonella encodes 5
T6SS loci, differentially distributed among different serotypes and with distinct
phylogenetic histories. The novel T6SS loci were identified in genomic islands
designated SPI-19, SPI-20, SPI-21 and SPI-22. In addition of the identification of these
T6SS loci, a novel “evolved” VgrG protein with a S-Type Pyocin containing-domain, was
identified in SPI-21. The presence of this protein domain suggested for the first time a
role for T6SSs in bacterial killing opening a new chapter in the study of T6SS and its
role in inter-bacterial relationships.
The SPI-19 T6SS was of significant relevance due to its wide distribution among
virulent Salmonella serotypes and because bioinformatics analyzes showed that while
Gallinarum encodes a complete T6SS, serotype Enteritidis only encodes for remnants
of this system. Despite being closely related, serotypes Gallinarum and Enteritidis
present profound differences in their host-range and pathogenicity. Therefore, it is
tempting to speculate that the presence of an active T6SS is somehow related to the
host-adaptation and pathogenicity differences presented by these serotypes. To resolve
this hypothesis and assess the contribution of SPI-19 to Salmonella pathogenicity, the
objective of this thesis was to determine whether the SPI-19 genomic island encodes a
functional T6SS contributing to the pathogenicity of Gallinarum and Enteritidis in the
avian host.
In order to characterize the SPI-19 T6SS, gene and operon fusions were
constructed and expression, production and secretion of T6SS components were
evaluated under different in vitro growth conditions. The analysis showed that most
T6SS components remain repressed under the conditions tested. Infection of murine
macrophages with a Gallinarum strain harboring the structural/secreted T6SS
component VgrG fused to the GFP reporter showed that T6SS components are preferentially produced inside infected cells. Non-polar deletion mutants of the whole
SPI-19 and specific T6SS core components revealed that this T6SS was necessary for
Salmonella Gallinarum survival within macrophages at late time points after infection.
Furthermore, the SPI-19 T6SS function could not be linked to Salmonella-induced
cytotoxicity or cell death of infected macrophages.
To determine the contribution of SPI-19 T6SS to Salmonella pathogenesis,
T6SS mutants were tested in competitive infection assays against the wild-type
Gallinarum parental strain. Oral infection of four-day-old White Leghorn chicks revealed
that T6SS mutants colonized the ileum, ceca, liver and spleen poorly compared to the
wild-type strain. Restitution of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture,
complemented this colonization defect. Altogether, the data indicate that SPI-19 and the
T6SS encoded therein contributes to macrophage intracellular survival and colonization
of chicks infected by S. Gallinarum. To assess the impact of carrying a complete T6SS
locus on the ability of S. Enteritidis to colonize the avian host, the SPI-19 from
Gallinarum was transferred to Enteritidis. In vivo experiments showed that presence of
a complete SPI-19 significantly increased the ability of Enteritidis to colonize the ileum,
liver and spleen of infected chicks by day 1 post-infection. This colonization advantage
was not lasting however, as this strain presented a strong colonization defect for each
organ analyzed from day 3 post infection to the conclusion of the experiment. These
results suggest that transfer of SPI-19 from S. Gallinarum has a negative impact on the
ability of S. Enteritidis to colonize the avian host. In this context is tempting to speculate
that loss of the SPI-19 T6SS corresponds to a pathoadaptative event during S.
Enteritidis evolution. In addition, this is the first report of the use of Vex-Capture method
to assess the effect of Genomic Island transfer in an animal model of bacterial infection.
The recent discovery of Type VI Secretion Systems (T6SS) has opened a new
chapter in the study of Salmonella host and environmental adaptation. Whether
Salmonella encodes T6SSs and whether they could be considered as quantum leap
evolution events are some of the questions that the discovery of T6SS in bacterial
genomes generated. In this thesis, we have expanded the current knowledge on
bacterial T6SSs and Salmonella virulence potential by: i) the identification and description of 4 novel Salmonella Pathogenicity Islands (SPI-19, SPI-20, SPI-21 and
SPI-22) encoding phylogenetically distinct T6SS loci, ii) the discovery of a novel
“evolved” VgrG protein, which suggested for the first time a role for T6SSs in interbacterial
relationships, iii) identifying that the SPI-19 T6SS contributes to Salmonella
intracellular survival in macrophages and iv) determining that the SPI-19 T6SS
contributes to chicken colonization by S. Gallinarum.
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Rol del sistema de secreción tipo seis (T6SS) codificado en la isla SPI-19 de Salmonella enterica serovares Enteritidis y Gallinarum, en la interacción con macrófagosJiménez Romaguera, Juan Cristóbal January 2010 (has links)
Salmonella Enteritidis y Salmonella Gallinarum son dos patógenos importantes para el hospedero aviar. El primero causa infecciones silentes en aves de postura, lo que le permite infectar los huevos y tras el consumo de éstos, llegar al hospedero humano. El segundo, causa en el ave un cuadro de gastroenteritis severa y posterior tifoidea aviar, generando graves consecuencias económicas y productivas. Previamente, mediante herramientas bioinformáticas, identificamos una nueva isla de patogenicidad no caracterizada en cuatro serotipos distintos de Salmonella, entre ellos Gallinarum y Enteritidis. Esta fue nombrada isla de patogenicidad 19 de Salmonella (SPI-19), y codificaría para un sistema de secreción tipo VI (T6SS), un sistema de secreción de proteínas el cual se encuentra ampliamente distribuido entre las bacterias Gram negativo. S. Gallinarum posee codificado en la SPI-19 todos los genes esenciales para un T6SS funcional, mientras que S. Enteritidis posee una versión trunca de la isla, y sólo posee algunos genes del T6SS. Este sistema cumpliría una serie de funciones en los procesos patogénicos dependiendo de la bacteria, y hasta el momento se ha descrito su participaciòn en adherencia, invasión, citotoxicidad y proliferación intracelular. Al analizar filogenéticamente el T6SS codificado en SPI-19 se descubrió que se encontraba cercano a los sistemas codificados en bacterias como Vibrio cholerae y Aeromonas hydrophila. En estos patógenos ya se ha descrito un rol del T6SS en la interacción con células del sistema inmune, particularmente en la sobrevida y citotoxicidad en líneas celulares de macrófagos. Debido a que la capacidad de Salmonella para sobrevivir dentro de macrófagos es un proceso fundamental para su diseminación sistémica y colonización de órganos blanco, en esta memoria se propuso determinar el papel que cumple el T6SS codificado en SPI-19 en la interacción con macrófagos murinos. Para esto se estudió la capacidad de sobrevivir al interior de macrófagos RAW 264.7 de mutantes en genes estructurales y efectores del T6SS. A su vez, se analizó la capacidad citotóxica de estas cepas. Los resultados muestran que mutantes de S. Gallinarum presentan una supervivencia menor a tiempos tardíos de infección. En S. Enteritidis la mutante para SPI-19 no presenta una disminución significativa de la sobrevida. En ambos serotipos no se presentan cambios en la citotoxicidad. También se estudió la expresión en la infección de macrófagos del efector VgrG mediante una fusión con el fluoróforo GFP. Esto reveló una rápida expresión de la fusión, lo que sugiere que el contacto bacteria-macrófago gatilla la expresión del efector. Estos resultados demuestran que la isla SPI-19 codifica para un T6SS funcional, que participa en un proceso esencial de la infección por Salmonella.
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Survival Strategies Of Salmonella Under Host Nitrosative Stress And Its Role In PathogenesisDas, Priyanka 08 1900 (has links)
Chapter: 1
Introduction
Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars belonging to Salmonella enterica. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Upon entry Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV). It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position.
Professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Upon activation of the inducible nitric oxide synthase (iNOS), NO is produced continuously at a high rate in the presence of adequate Larginine supply. Nitric oxide synthases catalyze the oxidation of one of the guanidino nitrogens of larginine to nitric oxide (NO). Of the multiple NOS isoforms that can catalyze NO synthesis, iNOS is mostly associated with antimicrobial activity. Host expression of iNOS is primarily regulated at the transcriptional level and can be stimulated following interaction with microbial products or in response to cytokines such as interleukin 1 (IL1), tumor necrosis factor α (TNFα) and interferon γ (IFNγ). To date, mutations that inactivate iNOS in humans have not been described. The importance of iNOS in human infection can therefore only be understood from indirect evidence and experimental models. Despite initial difficulty in demonstrating iNOS expression and NO production by human mononuclear phagocytes, an increasing body of evidence has identified a number of chronic inflammatory conditions, infectious diseases and in vitro treatments that stimulate iNOS mRNA expression and protein synthesis associated with NO bioactivity in human macrophages. Numerous studies have documented the production of RNIs in rodent models of Salmonella infection. Plasma nitrite and nitrate levels, a measure of RNI generation, have been shown to rise significantly after systemic infection of mice with S. Typhimurium.
Chapter:2
Role of nirC in Salmonella infection-Nitrosative stress response.
Activation of macrophages by interferon gamma (IFNγ) and the subsequent production of nitric oxide (NO) are critical for the host defense against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFNγ induced nitric oxide production in RAW264.7 macrophages infected with the wild type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from the IFNγ treated macrophages infected with the nirC mutant Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO dependent manner in the activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a 2fold more pronounced NO mediated repression of SPI2 was observed in the nirC knockout strain when compared to the wild type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT1 phosphorylation and iNOS expression than the wild type strain. In the iNOS knockout mice, the organ load of the nirC knockout strain was similar to the wild type strain indicating the fact that the mutant is exclusively sensitive towards the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade their killing in the activated macrophages by down regulating IFNγ induced NO production and highlights the critical role of nirC as a virulence gene.
Chapter:3
Salmonella mediated utilization of the host Arginine pool for intracellular growth -a novel strategy to survive.
Cationic amino acid transporters (CAT) are crucial regulators of both the nitric oxide synthase and arginase activity in the host cells as they regulate the Larginine availability. In this study, we show that Salmonella induces arginase activity in both the bone marrow derived macrophages and in dendritic cells in a LPS dependent manner. Further evidence is provided suggesting that the Salmonella mediated arginase induction is accompanied by an enhanced arginine uptake in the infected cells by up regulation of the expression of both mouse cationic amino acid transporters mCAT1 and mCAT2B. The bacterial growth was reduced in the presence of inhibitors of both arginase and arginine transport. We also observed that the argT knockout strain in Salmonella coding for an arginine permease was defective in the Larginine uptake and was also attenuated for growth in the mice model of infection. By utilizing both host and bacterial arginine transporters, Salmonella can access the host Larginine pool in the cytosol. The host CAT transporters co localize with the Salmonella containing vacuole in both the bone marrow derived macrophages and in dendritic cells. Thus the host arginine is channelized to the intracellular Salmonella for its growth and this novel strategy plays a pivotal role to counteract the stringent nutrient condition for the intracellular bacteria. On the other hand this channelization should ultimately decrease the substrate for NO production and serve as a survival strategy of the pathogenic Salmonella under host nitrosative stress.
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Characterization of the intracellular activities of SseJ and SifA, two Salmonella enterica serovar typhimurium type III secretion effector proteins /Ohlson, Maikke B. January 2007 (has links)
Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 112-125).
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